This proposal outlines an extension of earlier studies from our laboratory in murine tumor models which associated membrane transport differences in responsive tumors versus drug-limiting normal proliferative tissue with the selective action of the antifol, methotrexate. (1) A detailed study of structure-activity relationships for the general category of folate analogs is proposed to further define differences in the structural specificity of transport among a spectrum of sensitive and resistant murine tumors varying in responsiveness and normal mouse proliferative tissues. The structural specificity of dihydrofolate reductase and other key folate-related enzymes will also be sought in a comparative study. (2) The net pharmacologic expression of differences in membrane transport will be evaluated by pharmacokinetic measurements in each tissue following drug administration to the corresponding in vivo tumor model. (3) The pharmacologic/biochemical interrelationships in vivo which determine cytotoxicity, particularly with regard to the concentration-dependence for intracellular drug as well as a distinction between anti-thymidine versus antipurine effects will also be sought during animal experiments. The data from both in vitro (Aim 1) and in vivo (Aims 2 and 3) experiments will be applied to a more rational design of analogs with more differential interaction at membrane transport sites favoring greater effects against tumor. A new analog, 10-deazaaminopterin, with broad-spectrum antitumor activity in mice has already been identified in these studies and is scheduled for clinical trial. (4) Ongoing studies of high-dose methotrexate with leucovorin rescue have also yielded data now applied in the clinic. As a continuation of these studies, we plan to investigate aspects of the biochemical basis for differential rescue which these studies appear to suggest exists in tumor versus normal tissue.